The Cell Cycle as a Therapeutic Target for Cancer
Progression through the cell division cycle is controlled by oscillating waves of Cdk activity (1). These kinases are regulated positively by association with cyclin subunits and negatively by binding to Cdk inhibitors (2, 3). The Ubiquitin-Proteasome System (UPS) (FIG. 1) plays a key role in controlling cell cycle progression by promoting the periodic degradation of cyclins and Cdk inhibitors (4, 5).
Deregulation of cell cycle progression is a hallmark of human cancer (6). Although Cdks are rarely mutated in cancer, their activity is universally deregulated owing to hyperactivation of upstream signaling pathways (Ras-MAP kinase, PI 3-kinase), amplification of Cdk or cyclin genes, genetic/epigenetic inactivation of Ink4 Cdk inhibitors, or downregulation of p21 and p27 Cdk inhibitors (7-9). For example, cyclin D1 is overexpressed in several tumors as a result of transcriptional activation, gene amplification, or translocation. p16Ink4a a is frequently inactivated by gene deletion, point mutation or epigenetic silencing, resulting in activation of cyclin D-dependent kinases. Aberrant activation of Cdk2 and Cdk1 is observed in various malignancies. Other protein kinases such as Aurora A/B and Plk1, which are involved in centrosome duplication and mitosis execution, are overexpressed in a wide range of tumor types (10, 11). In addition to cell cycle kinases, deregulation of the mechanisms that control protein stability has been shown to contribute to tumorigenesis. Overexpression of oncogenic E3 ligases (such as Skp2), which target negative regulators of the cell cycle, or inactivation of tumor suppressor E3 ligases like Fbxw7 is observed in many human tumors (4, 5, 12).
Since it was established that aberrant cell cycle control is a hallmark of cancer, development of agents targeting the cell cycle has been viewed as a promising strategy for cancer therapy. For more than a decade, there has been an intensive search for small molecules that target Cdks, but no Cdk inhibitor drug has yet been approved for clinical use (7, 13, 14). More recent efforts have focused on the development of inhibitors for Aurora and Polo kinases (15-17). However, further investigation is necessary to assess the clinical potential of these targets. On the other hand, the FDA approval of the proteasome inhibitor bortezomib (Velcade; Millenium) for the treatment of multiple myeloma in 2003 (18) has heralded an entirely new class of cancer drugs and validated the therapeutic potential of the UPS (12, 19-22).
The Cip/Kip Family of Cdk Inhibitors
The activity of Cdks is negatively regulated by Cdk inhibitors. In human, 7 Cdk inhibitors have been identified and classified into two families, according to structural and functional similarities (1, 23). The Ink4 proteins, which include p16Ink4A, p15Ink4B, p18Ink4C and p19INK4D contain multiple ankyrin repeats and interact specifically with Cdk4 and Cdk6 to inactivate cyclin D-Cdk complexes. Members of the Cip/Kip family, which is composed of p21, p27 and p57, inhibit all cyclin-Cdk complexes and are not specific to a particular cell cycle phase. Structurally, the three Cip/Kip proteins share a conserved domain at their N-terminus, consisting of two separable subdomains for binding to cyclin and Cdk subunits (FIG. 2). They also have a nuclear localization signal (NLS) near the C-terminus. Notably, p21 also contains a proliferating cell nuclear antigen (PCNA) binding domain.
Biochemical and genetic analyses indicate that p21, p27 and p57 have both overlapping and specific cellular functions. p21 is a transcriptional target of p53 and is believed to be one of the main effectors of p53-mediated cell cycle arrest (24). The p21 protein is expressed ubiquitously in adult tissues. In the developing embryo, the expression of p21 correlates with terminal differentiation of a variety of tissues such as skeletal and heart muscle, cartilage and skin (25, 26). These observations implicated p21 in the regulation of cell cycle withdrawal during terminal differentiation. p27 is expressed ubiquitously and act as a negative regulator of cell proliferation in a variety of cell types (26). Accordingly, the expression of p27 is high in quiescent cells and in cells exposed to anti-proliferative signals, and declines in response to mitogenic factor stimulation (27-29). p57 is highly expressed in the developing embryo, but its expression declines in adults (26).
Regulation of p21 Expression in Normal and Cancer Cells
The regulation of p21 protein is exerted at multiple levels. The amount of p21 is controlled mainly at the levels of transcription and protein turnover (30). p21 was originally identified as the product of a gene activated by p53 (31). Since then, a variety of cellular and viral factors have been shown to induce or repress p21 transcription by p53-independent mechanisms (30, 32). In cancer cells, repression of p21 gene transcription is associated either with loss of function of activators (p53) or upregulation or gain of function mutations of transcriptional repressors. For example, the Myc oncogene is a potent repressor of p21 transcription (33). Importantly, p21 is a very unstable protein that is degraded by the proteasome (FIG. 3). Four E3 ubiquitin ligase complexes, SCFskp2 (34), CRL4cdt2 (35-37), APC/CCdc20 (38) and MKRN1 (39) have been shown to promote the degradation of p21 at specific stages of the cell cycle. Several proteins involved in the ubiquitin-dependent proteolysis of p21 are upregulated in a variety of human tumours, indicating that p21 downregulation may account for the oncogenic properties of these proteins. For example, Skp2, the substrate binding subunit of the SCFskp2 E3 ligase, is frequently upregulated in human cancers and displays oncogenic properties (4). Similarly, Cdt2 and Cul4a, two subunits of the CRL4cdt2 E3 ligase are overexpressed in breast and advanced liver cancers (40-43).
p21 is a Potent Tumor Suppressor
Mouse genetic studies and human clinical investigations have provided compelling evidence that p21 is a bona fide tumor suppressor. Mice deficient in p21 develop tumours of hematopoietic, endothelial and epithelial origin with late onset (44). Furthermore, p21 deficiency accelerates the development of chemically induced tumors in mice (45-47) and cooperates with oncogenes to promote tumorigenesis (48). Importantly, two recent studies have shown that knock-in mice expressing the p53 R172P mutant, that is deficient for apoptosis but maintains its ability to induce p21 and cell cycle arrest, are able to suppress tumorigenesis in different cancer models (49, 50). Tumor suppression by this p53 mutant was modulated by p21, which induced senescence and preserved chromosomal stability. p21 is not a classical tumor suppressor gene as it is very rarely mutated in human tumors. However, p21 levels are frequently downregulated in human cancers (including carcinomas, gliomas and hematological malignancies) and this is usually associated with a poor prognosis (30, 51). As mentioned above, downregulation of p21 is most often associated with increased turnover of the protein.
Accumulating evidence suggest that p21 exerts its tumor suppressor activity through multiple mechanisms. In addition to its ability to inhibit cyclin-Cdks and induce cell cycle arrest, microarray-based studies indicate that p21 expression is associated with the suppression of genes important for cell cycle progression and the induction of senescence genes (52). Interestingly, recent work suggests that tumor regression can be achieved through the reactivation of senescence, by restoring p53 function (53) or by inactivation of Myc in tumors with functional p53 (54). Reactivation of p53 and Myc inactivation both leads to p21 upregulation. p21 can compete for PCNA binding with several PCNA-reliant proteins involved in DNA repair processes (55). Finally, p21 has been reported to either inhibit or promote apoptosis depending on the cellular context (30). Interestingly, a recent study showed that p21 promotes apoptosis of intestinal stem/progenitor cells in response to gamma irradiation, suggesting that increasing p21 expression may be a viable approach to selectively target colon cancer stem cells (56).
There is thus a need for the development of novel strategies to inhibit p21 degradation, such as novel methods and assays to identify inhibitors of p21 degradation.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.